PROJECT SUMMARY This proposal is a renewal of 5R01EY017916-04 Mapping the optic lobes for color vision. Visual information gathered by the Drosophila eye is first processed in the optic lobes, lamina, medulla and lobula complex. The medulla is the first relay in the neural network for color vision, but it is the second step in the motion detection pathway. In the previous granting period, we have defined over 70 cell types that form overlapping retinotopic maps before projecting to the lobula complex. Because the medulla contains a finite number of neurons and connections that can be studied in exquisite detail, we can address fundamental questions about the development and function of a sophisticated neural structure. We present experiments to understand how optic neuron diversity is generated and how these neurons establish their retinotopic connections to photoreceptors. This will produce knowledge and tools that we will then use to analyze the function of individual neurons in optic pathways through electrophysiology and behavior assays. Three specific aims will help us reach these goals. (1). Sequential neuroblast (NB) switching generates neuronal diversity. We have discovered that the NBs generated as a wave of differentiation from the medulla neuroepithelium express sequentially at least three transcription factors in a process similar to the sequence observed in embryonic NBs. The neurons emerging from these NBs maintain expression of these genes and become different cell types. We will identify new NB and neuronal markers and identify the adult neuronal cell types derived from larval neurons marked by combinations of TFs. (2). Regionalization of medulla neuroepithelium and specialization of neuroblasts. The types of neurons generated by the sequentially generated NBs differ in distinct regions of the crescent shaped medulla Outer Proliferation Center (OPC). In the central part, young NBs produce both local columnar cells that remain where they were generated, as well as a smaller number of non-columnar neurons that migrate to occupy their retinotopic position in the entire adult medulla. We will analyze how the lineage of NBs is modified by their position along the OPC. We will then manipulate the positional identity of NBs and analyze the consequence on the neuronal composition of the medulla. We will thus define the combinatorial transcription factor code specifying medulla neuron types and will relate it to their adult fates. (3). Function of individual medulla neurons. We will record electrophysiological activity of these neurons in response to various light stimuli. We will continue our analysis of neurons involved in motion detection and extend this analysis of medulla neurons involved in chromatic pathways. The tools generated in aims 1 and 2 will allow us to silence of stimulate these neurons through 'intersectional' expression to analyze the motion and color behavior of flies using our flight simulator.